The steroid hormone (or nuclear) receptor superfamily has many members that play important roles in regulating cancer cells, including, but not limited to, the estrogen receptor (Bettuzi, 1991), the retinoic acid receptors (Menger, 1988), and v-erb A (Sharif, 1991). A number of less well characterized members of this family have been isolated and identified based on their homology to the better known nuclear receptors. (Laudet, 1992). Many show ligand-dependent activation patterns, but the identity of natural ligands that control these transcription factors is still unknown, or under investigation. Members of the steroid hormone receptor family whose ligands have not yet been identified are referred to as “orphan” receptors. The full importance of these “orphan” receptors is predicted by their presence early in development and by the fact that many (including PPAR, COUP, ROR) have been shown to interact with the known members of the superfamily (Beck, 1992; O'Malley, 1992; Tsukiyama, 1992). Many of these receptors are undoubtedly important in regulating cell growth and response to the environment, and may well have regulatory roles in cancer cells, like their currently well-studied counterparts.
The present invention provides a novel “orphan” receptor polypeptide, the human Peroxisome Proliferator Receptor γ (hPPAR-γ). It is a member of a group of orphan receptors found to be activated (but not bound) by arachidonic acid, fatty acids, clofibrate, and other agents that induce the proliferation of peroxisomes in rodents. (Blaauboer, 1990).
Peroxisome proliferators are a diverse group of chemicals which include hypolipidemic drugs, herbicides, leukotriene antagonists, and plasticizers. Two major categories of peroxisome proliferator chemicals play a significant role in society today. The first, the fibrate class of hypolipidemic drugs, has been found to be effective at reducing the levels of triglycerides and cholesterol in humans suffering from hyperlipidemia, a major risk factor for heart disease (Berioli, 1990). The second category relates to phthalate ester plasticizers used in the production of highly versatile flexible vinyl plastics (Reddy, 1983).
Peroxisome proliferators seem to affect most mammalian species that have been tested. They induce hepatomegaly resulting from liver hyperplasia and an increase in the size and number of peroxisomes. (Reddy, 1983.) Nevertheless, on the basis of hypolipidemic drug dose required to produce recognizable peroxisome proliferation, mice and rats are considered to be highly responsive to these agents, developing hepatocellular carcinoma following long-term drug administration; hamsters have intermediate responses; and guinea pigs, marmosets and other nonhuman primates are weakly responsive (Eacho, 1986).
Peroxisome proliferators are termed non-genotoxic carcinogens as they fail to cause DNA damage directly (Warren, 1980). The increase in peroxisomal fatty acid β-oxidation seen in response to peroxisome proliferators results in a greater production of hydrogen peroxide. It has been proposed that this results in oxidative stress leading to DNA damage and possible tumor initiation. (Reddy, 1983; Kasai, 1989). Alternatively, or in addition, peroxisome proliferators may act as liver-tumor promoters. (Marsman, 1988; Cattley, 1989).
The nuclear receptor subfamily to which the receptor polypeptide of the present invention belongs has been shown to regulate the transcription of several key enzymes in fatty acid metabolism, including Acyl Co A oxidase (Tugwood, 1992), and CYP4A6, a cytochrome P-450 omega hydroxylase, potentially having profound effects upon host responses via leukotriene and prostaglandin catabolism, fatty acid β oxidation, and superoxide production. Host response is a key aspect to the pathology of all diseases, including cancer, infection, and autoimmune disorders.
Recent studies demonstrate the PPARs can heterodimerize with the 9-cis retinoic acid receptor (RXR) and synergistically induce gene expression (Kliewer, 1992). These studies demonstrate a potential link between the retinoid responsive pathways and the lipid/arachidonic acid metabolic pathways. Preservation of the hydrophobic heptad repeats in the dimerization domain (see discussion below) indicates that PPARs can heterodimerize with other members of the thyroid/retinoid subbranch of the superfamily containing these repeats. There is also some conservation of the “conserved activating motif” (Daneilian et al., 1992) in the carboxy terminus.
PPARs are orphan receptors described in the murine (Issemann, 1990), rat (Gottlicher, 1992), human (Sher, 1993) and xenopus systems (Dreyer, 1992) as the peroxisome proliferator activated receptors α, β, and γ. A fourth member (h NUC 1) with some interesting unique sequence characteristics was isolated from human osteosarcoma cells (Schmidt, 1992). These receptors in other species are known to be activated by arachidonic acid at 150 μM levels, long chain fatty acids such as oleic and petroselenic acid, clofibrate, and other peroxisome proliferating agents (Gottlicher, 1992; Isseman, 1990). Arachidonic acid can trigger many different responses in cells such as neutrophils, but PPAR activation by long chain fatty acids provides evidence that arachidonic acid metabolites are candidate ligands or activators. In addition, murine PPAR-α has been shown to transactivate as a heterodimer with the human Retinoid X Receptor alpha (hRXR-α) both on retinoid receptor targets as well as on known PPAR target promoter sequences, potentially linking these pathways of gene regulation (Kliewer, 1992).
PPARs may play a role in proliferative and differentiation aspects of cancer, because they have also been shown to be developmentally active in vertebrates (xenopus), and are present in oocytes, fertilized eggs, blastulae, gastrulae, neurulae, and early tadpoles (Dreyer, 1992). It should be noted that many other members of the nuclear receptor superfamily appear to have two sets of regulatory functions. The first occurs during embryogenesis and development, including axis and pattern formation (Blumberg, 1992), and the second occurs in the adult, where the receptors modulate transcriptional activities in response to neighboring cells, other tissues, and aspects of the environment, particularly on regenerating tissues (Eider, 1991; Howell, 1990).
Members of the superfamily of nuclear hormone receptors have also been shown to directly connect the cellular transcriptional response to extracellular signals such as retinoids and steroids, as well as fatty acid and arachidonic acid metabolites, as already mentioned (Aronica, 1991). Studies of these receptors in hematopoiesis have primarily focused upon the retinoic acid receptor alpha (RAR-α) and v-erb A genes because of their important effects on hematopoietic cell differentiation.
The retinoic acid receptor α gene on chromosome 17 is translocated to chromosome 15 in virtually all cases of human acute promyelocytic leukemia (APL) (Rowley, 1988), where it is fused with PML gene. This generates two fusion proteins, containing RAR-α and PML sequences, which have different transcriptional activating properties than the wild type proteins. Treatment of APL patients with the ligand, all trans retinoic acid, consistently results in the achievement of a complete remission in this disease (Menger, 1988).
V-erb A is an aberrant version of a thyroid hormone receptor that can block erythroid differentiation and induce malignant transformation, an ability that is correlated with the repression of retinoic acid receptor function. Both retinoic acid receptors and v-erb A elicit significant effects upon the hematopoietic system by interacting with other transcripti on factors, such as fos and jun (Schule, 1991), and with other related nuclear receptors leading to heterodimer formation (Debois, 1991). The retinoic acid receptors, (RARs) can heterodimerize with retinoid x receptors (RXRs) in vitro, which increases their affinity for the RAR response element. RXRs can also heterodimerize with a number of different members of the superfamily in vitro, including the PPARs (Kliewer, 1992).
Target genes identified as transcriptionally activated by PPARs include acyl co-A oxidase, the key enzyme regulating the fatty acid B oxidation pathway (Tugwell, 1992), and CYP4A6, a cytochrome P450 fatty acid ω hydroxylase, a key enzyme catalyzing the ω hydroxylation of arachidonic, lauric and palmitic acids (Muerhoff, 1992). The sequence in the rat acyl-Co A oxidase promoter bound by PPARs is “acgTGACCTtTGTCCTggt” (SEQ ID NO:6) (Tugwell, 1992), which contains a direct repeat, separated by one nucleotide, (“DR-1” motif) (Umesono, 1991) of the canonical consensus half site “TGACCT” (SEQ ID NO:7) to which all members of the thyroid-retinoid branch of the nuclear receptor superfamily can bind (Laudet, 1992). The target sequences in CYP4A6 appear as imperfect DR-1 motifs and more complex arrangements of imperfect half sites. Candidate gene promoters must be tested for PPAR activation, but genes such as CD18, the leukocyte integrin β subunit, which has multiple combinations of imperfect half-sites and is retinoic acid inducible (Agura, 1992), are possibly PPAR responsive. Catalase, hydroxyacid oxidase, and uricase are down regulated by peroxisome proliferators in the rat, under the same conditions that up regulate acyl-coA oxidase. The effects of PPAR upon myeloperoxidase, chloroacetate esterase or catalase in myeloid cells have not been examined. Further characterization of these two forms of the PPAR-γ mRNA will be extremely useful for understanding their functions in hematopoietic cells as well as other organ systems.